Manufacturing of bupivacaine multivesicular liposomes

ABSTRACT

Embodiments of the present application relate to batches of bupivacaine multivesicular liposomes (MVLs) prepared by a commercial manufacturing process using independently operating dual tangential flow filtration modules.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

The present application is a continuation of U.S. application Ser. No.17/536,516, filed Nov. 29, 2021, which is a continuation of U.S.application Ser. No. 17/319,591, filed May 13, 2021, now U.S. Pat. No.11,185,506, which is a continuation of U.S. application Ser. No.17/156,400, filed Jan. 22, 2021, now U.S. Pat. No. 11,033,495, each ofwhich is incorporated by reference in its entirety.

BACKGROUND Field

This disclosure relates generally to commercial manufacturing processesfor making multivesicular liposomes using independently operatingtangential flow filtration systems.

Description of the Related Art

Bupivacaine is a versatile drug that has been shown to be efficaciousfor a wide variety of indications, including: local infiltration,peripheral nerve block, sympathetic nerve block, and epidural and caudalblocks. It may be used in pre-, intra- and post-operative care settings.Bupivacaine encapsulated multivesicular liposomes (Exparel®) has beenapproved in the US and Europe for use as postsurgical local analgesiaand as an interscalene brachial plexus nerve block to producepostsurgical regional analgesia, providing significant long-lasting painmanagement across various surgical procedures. Particularly, Exparel®has had great success in the market in part due to the ability tolocally administer bupivacaine multivesicular liposomes (MVLs) at thetime of surgery and extend the analgesic effects relative to othernon-liposomal formulations of bupivacaine. Such extended releaseproperties of bupivacaine MVLs allow patients to control theirpost-operative pain without or with decreased use of opioids. Given theaddictive nature of opioids and the opioid epidemic that has beenaffecting countries around the world, there is an urgent need for newand improved large scale productions of Exparel® to meet the substantialand growing market demand.

SUMMARY

Some aspects of the present disclosure relate to a crossflow filtrationsystem comprising:

a diafiltration vessel; and

a plurality of independently operating crossflow modules, each crossflowmodule of the plurality of independently operating crossflow modulescomprising at least one filter array, each filter array comprising aplurality of hollow fiber filters, wherein each crossflow module of theplurality of independently operating crossflow modules is connected to aretentate conduit, a permeate conduit, and a rotary lobe pump. In someembodiments, the crossflow filtration system may be used in themicrofiltration and/or diafiltration step of the commercial processdescribed herein.

Some aspects of the present disclosure relate to a process for preparingbupivacaine encapsulated multivesicular liposomes in a commercial scale,the process comprising:

(a) mixing a first aqueous solution comprising phosphoric acid with avolatile water-immiscible solvent solution to form a water-in-oil firstemulsion, wherein the volatile water-immiscible solvent solutioncomprises bupivacaine, at least one amphipathic lipid and at least oneneutral lipid;

(b) mixing the water-in-oil first emulsion with a second aqueoussolution to form a water-in-oil-in-water second emulsion;

(c) removing the volatile water-immiscible solvent from thewater-in-oil-in-water second emulsion to form a first aqueous suspensionof bupivacaine encapsulated multivesicular liposomes having a firstvolume;

(d) reducing the first volume of the first aqueous suspension ofbupivacaine encapsulated multivesicular liposomes by microfiltration toprovide a second aqueous suspension of bupivacaine encapsulatedmultivesicular liposomes having a second volume;

(e) exchanging the aqueous supernatant of the second aqueous suspensionwith a saline solution by diafiltration to provide a third aqueoussuspension of bupivacaine encapsulated multivesicular liposomes having athird volume; and

(f) further reducing the third volume of the third aqueous suspension bymicrofiltration to provide a final aqueous suspension of bupivacaineencapsulated multivesicular liposomes having a target concentration ofbupivacaine;

wherein all steps are carried out under aseptic conditions.

Some aspects of the present disclosure relate to a composition ofbupivacaine encapsulated multivesicular liposomes (MVLs) prepared by acommercial scale process, the commercial scale process comprising:

(a) mixing a first aqueous solution comprising phosphoric acid with avolatile water-immiscible solvent solution to form a water-in-oil firstemulsion, wherein the volatile water-immiscible solvent solutioncomprises bupivacaine, 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid;

(b) mixing the water-in-oil first emulsion with a second aqueoussolution to form a water-in-oil-in-water second emulsion;

(c) removing the volatile water-immiscible solvent from thewater-in-oil-in-water second emulsion to form a first aqueous suspensionof bupivacaine encapsulated MVLs having a first volume;

(d) reducing the first volume of the first aqueous suspension ofbupivacaine encapsulated MVLs by microfiltration to provide a secondaqueous suspension of bupivacaine encapsulated MVLs having a secondvolume;

(e) exchanging the aqueous supernatant of the second aqueous suspensionwith a saline solution by diafiltration to provide a third aqueoussuspension of bupivacaine encapsulated MVLs having a third volume; and

(f) further reducing the third volume of the third aqueous suspension bymicrofiltration to provide a final aqueous suspension of bupivacaineencapsulated MVLs having a target concentration of bupivacaine;

wherein all steps are carried out under aseptic conditions; and

wherein the erucic acid concentration in the composition is about 23μg/mL or less after the composition is stored at 25° C. for one month.

Some aspect of the present disclosure relates to a composition ofbupivacaine encapsulated multivesicular liposomes (MVLs), comprising:bupivacaine residing inside a plurality of internal aqueous chambers ofthe MVLs separated by lipid membranes, wherein the lipid membranescomprise 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid; and an aqueous medium in which the bupivacaineencapsulated MVLs are suspended; wherein the composition has an initialpH of about 7.0 to about 7.4, and wherein erucic acid concentration inthe composition is about 23 μg/mL or less after the composition isstored at 25° C. for one month.

Some additional aspect of the present disclosure relates to acomposition of bupivacaine encapsulated multivesicular liposomes (MVLs)prepared by a commercial scale process, the commercial scale processcomprising:

(a) mixing a first aqueous solution comprising phosphoric acid with avolatile water-immiscible solvent solution to form a water-in-oil firstemulsion, wherein the volatile water-immiscible solvent solutioncomprises bupivacaine, 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid;

(b) mixing the water-in-oil first emulsion with a second aqueoussolution to form a water-in-oil-in-water second emulsion, wherein thesecond aqueous solution comprises lysine and dextrose;

(c) removing the volatile water-immiscible solvent from thewater-in-oil-in-water second emulsion to form a first aqueous suspensionof bupivacaine encapsulated MVLs having a first volume;

(d) reducing the first volume of the first aqueous suspension ofbupivacaine encapsulated MVLs by microfiltration to provide a secondaqueous suspension of bupivacaine encapsulated MVLs having a secondvolume;

(e) exchanging the aqueous supernatant of the second aqueous suspensionwith a saline solution by diafiltration to provide a third aqueoussuspension of bupivacaine encapsulated MVLs having a third volume; and

(f) further reducing the third volume of the third aqueous suspension bymicrofiltration to provide a final aqueous suspension of bupivacaineencapsulated MVLs having a target concentration of bupivacaine;

wherein all steps are carried out under aseptic conditions; and

wherein the internal pH of the bupivacaine encapsulated MVLs in thecomposition is about 5.50. In some embodiments, the internal pH ismeasured after the composition has been stored at about 2-8° C. for atleast 3 months, 6 months or 9 months.

Some additional aspect of the present disclosure relates to acomposition of bupivacaine encapsulated multivesicular liposomes (MVLs),comprising: bupivacaine residing inside a plurality of internal aqueouschambers of the MVLs separated by lipid membranes, wherein the lipidmembranes comprise 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid; and an aqueous medium in which the bupivacaineencapsulated MVLs are suspended; wherein the internal pH of thebupivacaine encapsulated MVLs is about 5.50.

In any aspects of the disclosure described herein, the composition ofbupivacaine MVLs is suitable for human administration without furtherpurification.

BRIEF DESCRIPTION OF THE DRAWINGS

In addition to the features described above, additional features andvariations will be readily apparent from the following descriptions ofthe drawings and exemplary embodiments. It is to be understood thatthese drawings depict typical embodiments, and are not intended to belimiting in scope.

FIG. 1A illustrates a process flow chart of the formation of an initialaqueous suspension bupivacaine MVLs according to an embodiment of themanufacturing process described herein.

FIG. 1B illustrates a process flow chart of additional steps ofconcentration, filtration and solvent removal of the initial aqueoussuspension of bupivacaine MVLs according to an embodiment of themanufacturing process described herein.

FIG. 2 illustrate a crossflow filtration system according to anembodiment of the manufacturing process described herein.

FIG. 3A is a line chart showing supernatant pH as a function ofincubation time at 25° C. for bupivacaine-MVL compositions preparedaccording to a manufacturing process described herein as compared tobupivacaine-MVL compositions using the existing manufacturing process.

FIG. 3B is a line chart showing erucic acid concentration as a functionof incubation time at 25° C. for bupivacaine-MVL compositions preparedaccording to a manufacturing process described herein as compared tobupivacaine-MVL compositions prepared by the existing commercialmanufacturing process.

FIG. 3C is a chart showing erucic acid concentration as a function ofsupernatant pH at 25° C. for bupivacaine-MVL compositions preparedaccording to a manufacturing process described herein as compared tobupivacaine-MVL compositions prepared by the existing commercialmanufacturing process.

DETAILED DESCRIPTION

Embodiments of the present disclosure relate to new and improvedcommercial scale manufacturing processes for making bupivacaineencapsulated multivesicular liposomes (MVLs). The newly developedprocesses provide up to 5 folds increase in final product volume ascompared to the current process used for the manufacturing of Exparel®,which is disclosed in U.S. Pat. No. 9,585,838 and is incorporated byreference in its entirety. The processes also allow for improved productoperability. In addition, the improved and scaled up process also yieldsa more stabilized form of bupivacaine encapsulated MVLs, having lesslipid degradation byproducts, increased internal pH, and increasedlysine and dextrose encapsulation.

Definitions

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “bupivacaine encapsulated multivesicularliposomes”, “bupivacaine-MVLs” or “bupivacaine MVLs” refer to amultivesicular liposome composition encapsulating bupivacaine. In someembodiments, the composition is a pharmaceutical formulation, where thebupivacaine encapsulated multivesicular liposome particles are suspendedin a liquid suspending medium to form a suspension. In some suchembodiments, the BUP-MVL suspension may also include free orunencapsulated bupivacaine. In some cases, the free or unencapsulatedbupivacaine may be less than about 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.2% or 0.1%, by weight of the total amount of the bupivacaine in thecomposition, or in a range defined by any of the two preceding values.In some embodiment, the free bupivacaine may be about 5% or less byweight of the total amount of the bupivacaine in the composition. Infurther embodiments, the free bupivacaine may be about 8% or less duringthe shelf life of the product (i.e., up to 2 years when stored at 2-8°C.).

As used herein, the term “encapsulated” means that bupivacaine is insidea liposomal particle, for example, the MVL particles. In some instances,bupivacaine may also be on an inner surface, or intercalated in amembrane, of the MVLs.

As used herein, the term “unencapsulated bupivacaine” or “freebupivacaine” refers to bupivacaine outside the liposomal particles, forexample the MVL particles. For example, unencapsulated bupivacaine mayreside in the suspending solution of these particles.

As used herein, the term “median particle diameter” refers to volumeweighted median particle diameter of a suspension.

As used herein, a “pH adjusting agent” refers to a compound that iscapable of modulating the pH of an aqueous phase.

As used herein, the terms “tonicity” and “osmolality” are measures ofthe osmotic pressure of two solutions, for example, a test sample andwater separated by a semi-permeable membrane. Osmotic pressure is thepressure that must be applied to a solution to prevent the inward flowof water across a semi-permeable membrane. Osmotic pressure and tonicityare influenced only by solutes that cannot readily cross the membrane,as only these exert an osmotic pressure. Solutes able to freely crossthe membrane do not affect tonicity because they will become equalconcentrations on both sides of the membrane. An osmotic pressureprovided herein is as measured on a standard laboratory vapor pressureor freezing point osmometer.

As used herein, the term “sugar” as used herein denotes a monosaccharideor an oligosaccharide. A monosaccharide is a monomeric carbohydratewhich is not hydrolysable by acids, including simple sugars and theirderivatives, e.g. aminosugars. Examples of monosaccharides includesorbitol, glucose, fructose, galactose, mannose, sorbose, ribose,deoxyribose, dextrose, neuraminic acid. An oligosaccharide is acarbohydrate consisting of more than one monomeric saccharide unitconnected via glycosidic bond(s) either branched or in a chain. Themonomeric saccharide units within an oligosaccharide can be the same ordifferent. Depending on the number of monomeric saccharide units theoligosaccharide is a di-, tri-, tetra-, penta- and so forth saccharide.In contrast to polysaccharides, the monosaccharides and oligosaccharidesare water soluble. Examples of oligosaccharides include sucrose,trehalose, lactose, maltose and raffinose.

As used herein, the term “amphipathic lipids” include those having a netnegative charge, a net positive charge, and zwitterionic lipids (havingno net charge at their isoelectric point).

As used herein, the term “neutral lipid” refers to oils or fats thathave no vesicle-forming capabilities by themselves, and lack a chargedor hydrophilic “head” group. Examples of neutral lipids include, but arenot limited to, glycerol esters, glycol esters, tocopherol esters,sterol esters which lack a charged or hydrophilic “head” group, andalkanes and squalenes.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications referenced herein are incorporated by reference in theirentirety unless stated otherwise. In the event that there are aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise. As used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unlessotherwise indicated, conventional methods of mass spectroscopy, NMR,HPLC, protein chemistry, biochemistry, recombinant DNA techniques andpharmacology are employed. The use of “or” or “and” means “and/or”unless stated otherwise. Furthermore, use of the term “including” aswell as other forms, such as “include”, “includes,” and “included,” isnot limiting. As used in this specification, whether in a transitionalphrase or in the body of the claim, the terms “comprise(s)” and“comprising” are to be interpreted as having an open-ended meaning. Thatis, the terms are to be interpreted synonymously with the phrases“having at least” or “including at least.” When used in the context of aprocess, the term “comprising” means that the process includes at leastthe recited steps, but may include additional steps. When used in thecontext of a compound, composition, or device, the term “comprising”means that the compound, composition, or device includes at least therecited features or components, but may also include additional featuresor components.

Manufacturing Processes

Some embodiments of the present application relate to a commercial scalemanufacturing process for preparing bupivacaine encapsulatedmultivesicular liposomes. The process comprising:

(a) mixing a first aqueous solution comprising phosphoric acid with avolatile water-immiscible solvent solution to form a water-in-oil firstemulsion, wherein the volatile water-immiscible solvent solutioncomprises bupivacaine, at least one amphipathic lipid and at least oneneutral lipid;

(b) mixing the water-in-oil first emulsion with a second aqueoussolution to form a water-in-oil-in-water second emulsion;

(c) removing the volatile water-immiscible solvent from thewater-in-oil-in-water second emulsion to form a first aqueous suspensionof bupivacaine encapsulated multivesicular liposomes having a firstvolume;

(d) reducing the first volume of the first aqueous suspension ofbupivacaine encapsulated multivesicular liposomes by microfiltration toprovide a second aqueous suspension of bupivacaine encapsulatedmultivesicular liposomes having a second volume;

(e) exchanging the aqueous supernatant of the second aqueous suspensionwith a saline solution by diafiltration to provide a third aqueoussuspension of bupivacaine encapsulated multivesicular liposomes having athird volume; and

(f) further reducing the third volume of the third aqueous suspension bymicrofiltration to provide a final aqueous suspension of bupivacaineencapsulated multivesicular liposomes having a target concentration ofbupivacaine;

wherein all steps are carried out under aseptic conditions.

In some embodiments of the process, the amphipathic lipid in thevolatile water-immiscible solvent solution may be chosen from a widerange of lipids having a hydrophobic region and a hydrophilic region inthe same molecule. Suitable amphipathic lipids include, but are notlimited to zwitterionic phospholipids, including phosphatidylcholines,phosphatidylethanolamines, sphingomyelins, lysophosphatidylcholines, andlysophosphatidylethanolamines; anionic amphipathic phospholipids such asphosphatidylglycerols, phosphatidylserines, phosphatidylinositols,phosphatidic acids, and cardiolipins; cationic amphipathic lipids suchas acyl trimethylammonium propanes, diacyl dimethylammonium propanes,stearylamine, and the like. Non-limiting exemplary phosphatidyl cholinesinclude dioleyl phosphatidyl choline (DOPC), 1,2-dierucoylphosphatidylcholine or 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC),1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC),1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC),1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC),1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), or1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). Non-limitingexamples of phosphatidyl glycerols includedipalmitoylphosphatidylglycerol or1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG),1,2-dierucoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DEPG),1,2-dilauroyl-sn-glycero-3-phospho-rac-(1-glycerol) (DLPG),1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG),1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG),1,2-distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DSPG),1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG), orsalts thereof, for example, the corresponding sodium salts, ammoniumsalts, or combinations of the salts thereof. In some such embodiments,the amphipathic lipid comprises phosphatidylcholine, orphosphatidylglycerol or salts thereof, or combinations thereof. In someembodiments, the phosphatidyl choline is DEPC. In some embodiments, thephosphatidyl glycerol is DPPG. In some embodiments, the amphipathiclipid comprises DEPC and DPPG. In further embodiments, the DEPC and theDPPG are present in MVLs in a mass ratio of DEPC:DPPG of about 15:1 toabout 20:1, or about 17:1. In further embodiments, the total DEPC andDPPG in the MVLs suspension is in a mass ratio of about 7:1 to about10:1, or about 8:1.

In some embodiments, suitable neutral lipids in the volatilewater-immiscible solvent solution may include but are not limited totriglycerides, propylene glycol esters, ethylene glycol esters, andsqualene. Non-limiting exemplary triglycerides useful in the instantformulations and processes are triolein (TO), tripalmitolein,trimyristolein, trilinolein, tributyrin, tricaproin, tricaprylin (TC),and tricaprin. The fatty acid chains in the triglycerides useful in thepresent application can be all the same, or not all the same (mixedchain triglycerides), or all different. In one embodiment, the neutrallipid comprises or is tricaprylin. In further embodiments, the volatilewater-immiscible solvent solution in step (a) of the process may furthercomprise cholesterol and/or a plant sterol.

In some embodiments of the process described herein, the mixing in step(a) is performed using a first mixer at a high shear speed. In someembodiments, the high shear speed is from about 1100 rpm to about 1200rpm, for example, 1100 rpm, 1110 rpm, 1120 rpm, 1130 rpm, 1140 rpm, 1150rpm, 1160 rpm, 1170 rpm, 1180 rpm, 1190 rpm, or 1200 rpm, or a rangedefined by any of the two preceding values. In some embodiment, the highshear speed is about 1150 rpm. In some embodiments, the mixing in step(a) is performed for about 65 minutes, 66 minutes, 67 minutes, 68minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74minutes or 75 minutes. Proper mixing rate is important for forming thefirst emulsion droplets in a proper size range, which is important tothe final product yield, the MVL particle stability and releaseproperties. It was observed that when the mixing speed is too low or toohigh, the droplets formed in the first emulsion were either too big ortoo small. In some further embodiments, the first mixer used in step (a)of the process has a blade diameter of between about 8 inch to about 10inch. In further embodiments, the first mixer used in step (a) of theprocess is not a static mixer. In further embodiments, the mixing instep (a) is performed at a temperature of about 21° C. to about 23° C.

In some embodiments of the process described herein, the mixing in step(b) is performed using a second mixer at a low shear speed. In someembodiments, the low shear speed is from about 450 rpm to about 510 rpm,for example, 450 rpm, 455 rpm, 460 rpm, 465 rpm, 470 rpm, 475 rpm, 480rpm, 485 rpm, 490 rpm, 495 rpm, 500 rpm, 505 rpm, or 510 rpm, or a rangedefined by any of the two preceding values. In some embodiment, the lowshear speed is about 495 rpm. In some embodiments, the mixing in step(b) is performed for about 60 seconds, 61 seconds, 62 seconds, 63seconds, 64 seconds, or 65 seconds. In some further embodiments, thesecond mixer used in step (b) of the process has a blade diameter ofbetween about 10 inch to about 15 inch, for example, 10 inch, 11 inch,12 inch, 13 inch, or 14 inch. In further embodiments, the second mixerused in step (b) of the process is not a static mixer. In furtherembodiments, the mixing in step (b) is performed at a temperature ofabout 21° C. to about 23° C. The water-in-oil-in water (w/o/w) secondemulsion is not as stable as the first emulsion. As such, a low shearspeed was used in mixing step to reduce the disruption of the spherulesformed in this step. In addition, the mixing time in step (b) is alsoimportant to yield the final MVL particles in the target diameters andhave the desired release properties. If mixing time is too short, it ledto a larger particle size.

In some embodiments of the process described herein, the second aqueoussolution comprises one or more pH modifying agents. The pH modifyingagents that may be used in the present MVL formulations are selectedfrom organic acids, organic bases, inorganic acids, or inorganic bases,or combinations thereof. Suitable organic bases that can be used in thepresent application include, but are not limited to histidine, arginine,lysine, tromethamine (Tris), etc. Suitable inorganic bases that can beused in the present application include, but are not limited to sodiumhydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide,etc. Suitable inorganic acids (also known as mineral acids) that can beused in the present application include, but are not limited tohydrochloric acid (HCl), sulfuric acid (H₂SO₄), phosphoric acid (H₃PO₄),nitric acid (HNO₃), etc. Suitable organic acids that can be used in thepresent application include, but are not limited to acetic acid,aspartic acid, citric acid, formic acid, glutamic acid, glucuronic acid,lactic acid, malic acid, tartaric acid, etc. In one embodiment, the pHmodifying agent comprises lysine.

In some embodiments of the process described herein, the second aqueoussolution comprises one or more tonicity agents. Tonicity agentssometimes are also called osmotic agents. Non-limiting exemplary osmoticagents suitable for the MVL formulation of the present applicationinclude monosaccharides (e.g., glucose, and the like), disaccharides(e.g., sucrose and the like), polysaccharide or polyols (e.g., sorbitol,mannitol, Dextran, and the like), or amino acids. In some embodiments,one or more tonicity agents may be selected from an amino acid, a sugar,or combinations thereof. In some further embodiments, one or moretonicity agents are selected from dextrose, sorbitol, sucrose, lysine,or combinations thereof. In one embodiment, the tonicity agent comprisesdextrose. In some further embodiments, the second aqueous solutioncomprises lysine and dextrose.

In some embodiments of the process described herein, the volatilewater-immiscible organic solvent comprises or is methylene chloride(CH₂C₁₂). The organic solvent is substantially removed by exposing thesecond emulsion to a gas atmosphere. Organic solvent may be removed byblowing a gas over the second emulsion, or sparging gas in the secondemulsion, or spraying the second emulsion into a chamber with acontinuous stream of circulating gas.

In some embodiments of the process described herein, wherein step (e) isperformed using two sets of filtration modules, wherein each set of thefiltration modules operate independently of the other. In furtherembodiments, each set of the filtration module comprises five or morehollow fiber filters, each having a membrane pore size from about 0.1 μmto about 0.2 μm. One embodiment of the filtration modules is illustratedin FIG. 2 .

In some embodiments of process described herein, the diafiltration step(e) is performed multiple times until the aqueous supernatant of thesecond aqueous suspension is substantially replaced with the salinesolution.

In some embodiments of process described herein, step (f) may beperformed multiple times until a target concentration of bupivacaineMVLs is reached. In some further embodiments, the final aqueoussuspension of bupivacaine encapsulated multivesicular liposomes istransferred to a bulk product vessel.

FIGS. 1A-1B are process flow charts, each depicting a portion of thebupivacaine MVLs manufacturing process 100 according to some embodimentsdescribed herein. The circled A symbol indicates the connection pointbetween FIG. 1A and FIG. 1B. As shown in FIGS. 1A-1B, bupivacaine MVLsis produced via an aseptic double-emulsion process. The bulkmanufacturing system is a closed, sterilized system into which allprocess solutions are sterile-filtered through 0.2 μm filters.

As shown in FIG. 1A, the process 100 includes a step 102, wherein DEPC,DPPG, cholesterol, tricaprylin, and bupivacaine are dissolved inmethylene chloride to form a lipid/drug solution 102. At a step 103, thelipid solution is filtered through a 0.2 μm membrane filter into asterilized vessel. At a step 104, phosphoric acid is dissolved in WFI(water for injection) to form a H₃PO₄ solution. At a step 105, the H₃PO₄solution is filtered through a 0.2 μm membrane filter into a sterilizedvessel. Under aseptic conditions, the filtered lipid/drug solution iscombined with the filtered H₃PO₄ solution in a volume ratio of 1:1 at anemulsification step 106 using agitation to produce a w/o emulsion (i.e.,first emulsion). High shear mixing of the lipid/drug solution with thephosphoric acid solution is performed, wherein bupivacaine is ionized bythe phosphoric acid and partitions into the internal aqueous phase. At astep 107, lysine and dextrose are combined in WFI to form adextrose/lysine solution. At a step 108, the dextrose/lysine solution isfiltered through a 0.2 μm membrane filter into a sterilized vessel.Under aseptic conditions, the filtered dextrose/lysine solution is addedto the w/o emulsion in a volume ratio of approximately 2.5:1 at anemulsification step 109 using agitation to produce a w/o/w emulsion(i.e., second emulsion). At emulsification step 109, agitation isperformed at lower shear, producing a water-in-oil-in-water (w/o/w)emulsion with the majority of the bupivacaine resident in the internalaqueous phase. Additional filtered dextrose/lysine solution is added tothe w/o/w emulsion at a dilution step 110 to form a diluted suspensionof MVLs and bringing the final volume ratio to approximately 20:1(dextrose/lysine solution to water-in-oil emulsion) with mixing. At astep 111, the diluted suspension of MVLs is sparged with sterilenitrogen to remove the majority of the methylene chloride.

FIG. 1B depicts additional steps of the process 100. After sparging atstep 111, the diluted suspension of bupivacaine MVLs is concentrated viaaseptic microfiltration at a step 112 to a bupivacaine concentration ofapproximately 4.5 mg/mL.

At a step 113, a NaCl solution is formed by dissolving sodium chloridein WFI. At a step 114, the NaCl solution (i.e., saline solution) isfiltered through a 0.2 μm membrane filter. Under aseptic conditions, thebupivacaine MVLs concentrate formed at step 112 is subjected tocrossflow filtration by at least four volumes of the filtered NaClsolution through introduction of the filtered NaCl solution into acrossflow filtration apparatus or system through multiple 0.2 μm hollowfiber filter membrane unit filters at a diafiltration step 115.Diafiltration step 115 is used to remove unencapsulated bupivacaine,lysine, dextrose and residual methylene chloride, thereby reducing thesuspension volume and increasing the concentration of the bupivacaineMVLs in the suspension. At a step 116, sterile nitrogen is used to flushthe headspace of the crossflow filtration apparatus or system to furtherreduce residual methylene chloride content. The solution is furtherconcentrated via aseptic microfiltration in concentrate step 117 to forman initial bulk suspension of MVLs at a target weight/volume thatcorresponds to a bupivacaine concentration of 11.3-16.6 mg/mL. The bulkproduct is then transferred into a sterilized holding vessel. Theinitial bulk suspension of MVLs is sampled and bupivacaine concentrationis measured. Optionally, if the initial bulk suspension of MVLs isdesignated to be filled as an individual lot, the initial bulksuspension of MVLs is concentrated further via sedimentation(gravitational settling) and/or decantation to a bupivacaineconcentration of approximately 13.3 mg/mL, or alternatively diluted witha filtered NaCl solution to a bupivacaine concentration of approximately13.3 mg/mL at a decantation and/or dilution step 120 to form an adjustedbulk suspension of MVLs. The saline solution that is optionally used atstep 120 can be formed by dissolving sodium chloride in WFI at a step118 and filtered through a 0.2 μm membrane filter at a step 119.

Tangential Flow Filtration Modules

Some embodiments of the present application relates to a crossflowfiltration system comprising: a diafiltration vessel; and a plurality ofindependently operating crossflow modules, each crossflow module of theplurality of independently operating crossflow modules comprising atleast one filter array, each filter array comprising a plurality ofhollow fiber filters, wherein each crossflow module of the plurality ofindependently operating crossflow modules is connected to a retentateconduit, a permeate conduit, and a rotary lobe pump. In someembodiments, the crossflow filtration system may be used in themicrofiltration and/or diafiltration step of the commercial processdescribed herein.

In some embodiments, each crossflow module comprises two filter arrays.In some embodiments, each crossflow module comprises at least fivehollow fiber filters. In some such embodiments, each filter arraycomprises at least two hollow fiber filters.

In some embodiments, the plurality of independently operating crossflowmodules comprises a first crossflow module and a second crossflowmodule, wherein the first crossflow module is coupled to a first rotarylobe pump and the second crossflow module is coupled to a second rotarylobe pump operating independently of the first rotary lobe pump. In somefurther embodiments, the first crossflow module is coupled to thediafiltration vessel by a first retentate conduit to facilitate flow ofretentate from the first crossflow module to the diafiltration vessel,and wherein the second crossflow module is coupled to the diafiltrationvessel by a second retentate conduit to facilitate flow of retentatefrom the second crossflow module to the diafiltration vessel. In somefurther embodiments, the first rotary lobe pump comprises a fluid outletcoupling the first rotary lobe pump to the first crossflow module, andwherein the second rotary lobe pump comprises a fluid outlet couplingthe second rotary lobe pump to the first crossflow module. In somefurther embodiments, the first rotary lobe pump comprises a fluid inletcoupling the first rotary lobe pump to the diafiltration vessel, andwherein the second rotary lobe pump comprises a fluid inlet coupling thesecond rotary lobe pump to the diafiltration vessel.

In some embodiments, the first crossflow module operates independentlyfrom the second crossflow module. In some such embodiments, only one ofthe first crossflow module and the second crossflow module is in useduring the operation of the crossflow filtration system. In otherembodiments, both the first crossflow module and the second crossflowmodule are in use during the operation of the crossflow filtrationsystem.

In some embodiments, each of the plurality of independently operatingcrossflow modules comprises a microfiltration mode and a diafiltrationmode.

In some embodiments, the crossflow filtration system further comprises anitrogen sparging module to blow a stream nitrogen over the retentate inthe diafiltration vessel.

Some further embodiments of the present application relate to a processof manufacturing bupivacaine encapsulated multivesicular liposomes usingthe cross-flow module described herein, the process comprising:

reducing a first aqueous suspension of bupivacaine encapsulated MVLshaving a first volume by microfiltration to provide a second aqueoussuspension of bupivacaine encapsulated MVLs having a second volume;

exchanging the aqueous supernatant of the second aqueous suspension witha saline solution by diafiltration to provide a third aqueous suspensionof bupivacaine encapsulated MVLs having a third volume; and furtherreducing the third volume of the third aqueous suspension bymicrofiltration to provide a final aqueous suspension of bupivacaineencapsulated MVLs having a target concentration of bupivacaine.

In some embodiments, the process further comprises blowing a stream ofnitrogen over the second aqueous suspension during thediafiltration/saline exchange step. In some further embodiments, thediafiltration include at least two, three, four or five exchange volumesof the saline solution such that the aqueous supernatant of the secondaqueous suspension is substantially (e.g., at least 95%, 96%, 97%, 98%,99%) replaced by the saline solution.

Some further embodiments relate to a composition of bupivacaineencapsulated multivesicular liposomes prepared by the process utilizingthe crossflow filtration system described herein.

FIG. 2 depicts an embodiment of a crossflow filtration system 200 foruse in a diafiltration step of a commercial scale manufacturing processas described herein, such as step 115 of the process 100. The system 200includes independently operating crossflow modules 202 a and 202 b.Crossflow module 202 a includes a filter array 204 a and a filter array204 b. Crossflow module 202 b includes a filter array 204 c and a filterarray 204 d. Each filter array 204 a-d may include two or more hollowfiber filters. In some embodiments, each filter array includes five ormore hollow fiber filters.

The system may be connected to a sparge/diafiltration vessel 206.Retentate can flow from the crossflow module 202 a to the vessel 206 viaa retentate return conduit 208 a. Retentate can flow from the crossflowmodule 202 b to the vessel 206 via a retentate return conduit 208 b.Permeate can flow from the crossflow module 202 a for removal from thesystem 200 via a permeate conduit 210 a. Permeate can flow from thecrossflow module 202 b for removal from the system 200 via a permeateconduit 210 b.

The system 200 may include or be used in conjunction with twoindependently operating rotary lobe pumps 212 a and 212 b. The pump 212a includes a fluid inlet 214 a and a fluid outlet 216 a. The pump 212 bincludes a fluid inlet 214 b and a fluid outlet 216 b. The pump 212 a isconnected to the vessel 206 via the inlet 214 a and connected tocrossflow module 202 a via the outlet 216 a. The pump 212 b is connectedto the vessel 206 via the inlet 214 b and connected to the crossflowmodule 202 b via the outlet 216 b.

In some embodiments of the process described herein, the crossflowfiltration system utilizes two independent rotary lobe pumps providingretentate flow to independent arrays of five hollow fiber filterhousings. This configuration allows for smaller pipe diameters to allowfor turbulent flow. In addition, the filtration module design allows fortwo filter arrays to be in-use during bulk operation while two filterarrays are being cleaned and sterilized in preparation for the next bulkproduction run. This configuration allows for shorter cycle times andincreased manufacturing capacity. Furthermore, the improved filtrationmodule design allows for independent hollow fiber filter housingisolation. This functionality automatically detects and isolatesindividual filter integrity failures, allowing the bulk cycle to proceedwithout offline testing and recleaning. In some further embodiments, theprocess may further comprise an additional product recovery step fromone of the two filter array and/or a saline flush step, to allow fornearly complete product recover from the transfer lines and therebyincreasing product yield.

In some embodiments of the process described herein, the final aqueoussuspension of bupivacaine encapsulated multivesicular liposomes has avolume of about 150 L to about 250 L. In one embodiment, the finalaqueous suspension of bupivacaine encapsulated multivesicular liposomeshas a volume of about 200 L. In another embodiment, the final aqueoussuspension of bupivacaine encapsulated multivesicular liposomes has avolume of about 225 L. In some embodiments, the percent packed particlevolume (% PPV) of the final aqueous suspension of bupivacaineencapsulated multivesicular liposomes is about 32% to about 44%, about35% to about 40%, or about 36% to about 38%. In some such embodiments,the target concentration of the bupivacaine in the final aqueoussuspension (i.e., bulk product suspension) is from about 12.6 mg/mL toabout 17 mg/mL. In further embodiments, the final product targetconcentration of the bupivacaine in the aqueous suspension is about 13.3mg/mL. In some embodiments, the final aqueous suspension of bupivacaineMVLs comprises less than 5%, 4%, 3%, 2% or 1% unencapsulatedbupivacaine, wherein the amount of unencapsulated bupivacaine iscalculate based on the total weight of the bupivacaine in the aqueoussuspension. In some embodiments, the d₅₀ of the multivesicular liposomesin the final aqueous suspension is about 24 μm to about 31 μm. In oneembodiment, the d₅₀ of the multivesicular liposomes in the final aqueoussuspension is about 27 μm. In some embodiments, the internal pH of thebupivacaine encapsulated multivesicular liposomes is about 5.5. In somesuch embodiments, the lysine concentration inside the bupivacainemultivesicular liposome particles (i.e., internal lysine concentrationor encapsulated lysine concentration) is about 0.08 mg/mL. In furtherembodiments, the internal lysine concentration is about 0.03 mg/mL,where the lysine concentration is measured when the MVL particles are inthe aqueous suspension. In some embodiments, the external pH of thebupivacaine encapsulated multivesicular liposomes is about 7.0 to about7.4. As used herein, “internal pH” of the bupivacaine MVLs refer to thepH of the internal aqueous chambers of the MVL particles. The pH of theaqueous suspension of the bupivacaine MVLs is also referred to as the“external pH” of the bupivacaine MVLs. In some embodiments, the externalpH of the bupivacaine MVLs are measured during the product's shelf lifeunder the storage condition between 2-8° C. When the bupivacaine MVLsare stored at ambient temperature at extended period of time, theexternal pH of the composition may drop below the 7.0-7.4 rangepartially due to the accelerated lipid hydrolysis.

Bupivacaine Multivesicular Liposomes Prepared by the New Process

MVLs are a group of unique forms of synthetic membrane vesicles that aredifferent from other lipid-based delivery systems such as unilamellarliposomes and multilamellar liposomes (Bangham, et al., J Mol. Bio.,13:238-252, 1965). The main structural difference between multivesicularliposomes and unilamellar liposomes (also known as unilamellar vesicles,“ULVs”), is that multivesicular liposomes contain multiple aqueouschambers per particle. The main structural difference betweenmultivesicular liposomes and multilamellar liposomes (also known asmultilamellar vesicles, “MLVs”), is that in multivesicular liposomes themultiple aqueous chambers are non-concentric. Multivesicular liposomesgenerally have between 100 to 1 million chambers per particle and allthe internal chambers are interconnected by shared lipid-bilayer wallsthat separate the chambers. The presence of internal membranesdistributed as a network throughout multivesicular liposomes may serveto confer increased mechanical strength to the vesicle. The particlesthemselves can occupy a very large proportion of the total formulationvolume. Such formulation is intended to prolong the local delivery ofbupivacaine, thereby enhancing the duration of action of the reductionof pain.

The bupivacaine MVLs produced by the process described herein haveimproved stability over the commercial Exparel® product. It was observedthat the bupivacaine MVL particles produced by the process describedherein have lower lipid hydrolysis byproducts compared to the commercialExparel® product under the same incubation condition. In addition, thebupivacaine MVL particles produced by the process described herein hashigher internal lysine and dextrose concentrations and more desirableinternal pH, which may improve MVL particle strength during producttransportation, as well as lipid membrane stability.

Some embodiments of the present disclosure relate to a composition ofbupivacaine encapsulated multivesicular liposomes (MVLs) prepared by acommercial scale process described herein, the commercial scale processcomprising:

(a) mixing a first aqueous solution comprising phosphoric acid with avolatile water-immiscible solvent solution to form a water-in-oil firstemulsion, wherein the volatile water-immiscible solvent solutioncomprises bupivacaine, 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid;

(b) mixing the water-in-oil first emulsion with a second aqueoussolution to form a water-in-oil-in-water second emulsion;

(c) removing the volatile water-immiscible solvent from thewater-in-oil-in-water second emulsion to form a first aqueous suspensionof bupivacaine encapsulated MVLs having a first volume;

(d) reducing the first volume of the first aqueous suspension ofbupivacaine encapsulated MVLs by microfiltration to provide a secondaqueous suspension of bupivacaine encapsulated MVLs having a secondvolume;

(e) exchanging the aqueous supernatant of the second aqueous suspensionwith a saline solution by diafiltration to provide a third aqueoussuspension of bupivacaine encapsulated MVLs having a third volume; and(f) further reducing the third volume of the third aqueous suspension bymicrofiltration to provide a final aqueous suspension of bupivacaineencapsulated MVLs having a target concentration of bupivacaine;

wherein all steps are carried out under aseptic conditions; and whereinthe erucic acid concentration in the composition is about 23 μg/mL orless after the composition is stored at 25° C. for one month. In oneembodiment, the erucic acid concentration in the composition is about22.7 μg/mL after the composition is stored at 25° C. for one month.

In some embodiments, the final aqueous suspension of bupivacaineencapsulated MVLs described in the process is the composition of thebupivacaine MVLs described herein. In other embodiments, theconcentration of the final aqueous suspension of bupivacaineencapsulated MVLs described in the process may be further adjusted witha saline solution to provide the composition of the bupivacaine MVLsdescribed herein. In some embodiments, the composition has a pH of about7.1 after the composition is stored at 25° C. for one month.

In some further embodiments, the erucic acid concentration in thecomposition is about 38 μg/mL or less after the composition is stored at25° C. for two months. In one embodiment, the erucic acid concentrationin the composition is about 37.3 μg/mL after the composition is storedat 25° C. for two months. In some such embodiments, the composition hasa pH of about 7.1 after the composition is stored at 25° C. for twomonths.

In some further embodiments, the erucic acid concentration in thecomposition is about 54 μg/mL or less after the composition is stored at25° C. for three months. In one embodiment, the erucic acidconcentration in the composition is about 53 μg/mL after the compositionis stored at 25° C. for three months. In some such embodiments, thecomposition has a pH of about 6.9 after the composition is stored at 25°C. for three months.

In some further embodiments, the erucic acid concentration in thecomposition is about 100 μg/mL or less after the composition is storedat 25° C. for six months. In one embodiment, the erucic acidconcentration in the composition is about 98.7 μg/mL after thecomposition is stored at 25° C. for six months. In some furtherembodiments, the composition has a pH of about 6.5 after the compositionis stored at 25° C. for six months.

In some embodiments, the composition of bupivacaine MVLs comprises thefollowing lipid components: DEPC, DPPG, cholesterol and tricaprylin. Insome embodiments, the total concentrations of the lipid components inthe composition are the following: DEPC (about 7.0 mg/mL), DPPG (about0.9 mg/mL), cholesterol (about 4.2 mg/mL), tricaprylin (about 1.6mg/mL). Since DEPC has the highest concentration of all the lipidcomponents, the hydrolysis byproduct of DEPC is used as the marker toassess lipid stability of the MVLs. The hydrolysis byproducts of DEPCinclude erucic acid and lyso-DEPC (1- and 2-isomers). Lyso-DEPC isformed by hydrolysis of DEPC. Lyso-DEPC can further hydrolyze toglycerophosphocholine and erucic acid. In some embodiments, the erucicacid concentration in the composition of bupivacaine MVLs produced bythe process described herein is at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% less than the erucic acidconcentration in the Exparel® product manufactured by the currentcommercial process, under the same incubation condition. In some suchembodiments, the incubation condition is at 25° for 1 month, 2 months, 3months, or 6 months.

Some additional embodiments of the present disclosure relate to acomposition of bupivacaine encapsulated multivesicular liposomes (MVLs)prepared by a commercial scale process described herein, the commercialscale process comprising:

(a) mixing a first aqueous solution comprising phosphoric acid with avolatile water-immiscible solvent solution to form a water-in-oil firstemulsion, wherein the volatile water-immiscible solvent solutioncomprises bupivacaine, 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid;

(b) mixing the water-in-oil first emulsion with a second aqueoussolution to form a water-in-oil-in-water second emulsion, wherein thesecond aqueous solution comprises lysine and dextrose;

(c) removing the volatile water-immiscible solvent from thewater-in-oil-in-water second emulsion to form a first aqueous suspensionof bupivacaine encapsulated MVLs having a first volume;

(d) reducing the first volume of the first aqueous suspension ofbupivacaine encapsulated MVLs by microfiltration to provide a secondaqueous suspension of bupivacaine encapsulated MVLs having a secondvolume;

(e) exchanging the aqueous supernatant of the second aqueous suspensionwith a saline solution by diafiltration to provide a third aqueoussuspension of bupivacaine encapsulated MVLs having a third volume; and

(f) further reducing the third volume of the third aqueous suspension bymicrofiltration to provide a final aqueous suspension of bupivacaineencapsulated MVLs having a target concentration of bupivacaine;

wherein all steps are carried out under aseptic conditions; and

wherein the internal pH of the bupivacaine encapsulated MVLs in thecomposition is about 5.50. In some embodiments, the internal pH ismeasured after the composition has been stored at about 2-8° C. for atleast 3 months, 6 months or 9 months. In one embodiment, the internal pHis measured after the composition has been stored at about 2-8° C. forabout 9 months.

In some embodiments, the final aqueous suspension of bupivacaineencapsulated MVLs described in the process is the composition of thebupivacaine MVLs described herein. In other embodiments, theconcentration of the final aqueous suspension of bupivacaineencapsulated MVLs described in the process may be further adjusted witha saline solution to provide the composition of the bupivacaine MVLsdescribed herein.

In some embodiments of the composition described herein, the lysineconcentration inside the bupivacaine encapsulated MVL particles of thecomposition (internal lysine concentration or encapsulated lysineconcentration) is about 0.030 mg/mL to about 0.032 mg/mL. In some suchembodiment, the internal lysine concentration is measured when thebupivacaine MVLs are suspended in an aqueous suspension with % PPV about36.5%. In some further embodiments, the internal lysine concentrationinside the bupivacaine encapsulated MVL particles is about 0.031 mg/mL.

In some embodiments of the composition described herein, the dextroseconcentration inside the bupivacaine encapsulated MVL particles of thecomposition (internal dextrose concentration or encapsulated dextroseconcentration) is about 1.25 mg/mL to about 1.32 mg/mL. In some suchembodiment, the internal dextrose concentration is measured when thebupivacaine MVLs are suspended in an aqueous suspension with % PPV about36.5%. In some further embodiments, the internal dextrose concentrationinside the bupivacaine encapsulated MVL particles is about 1.29 mg/mL.

Although it is expected that only phosphoric acid resides inside theinternal aqueous chambers of the MVL particles (i.e., the first emulsionis formed by mixing the phosphoric acid aqueous solution with a volatilewater-immiscible solvent solution). However, there are also very smallamounts of lysine and dextrose encapsulated inside the internal aqueouschambers during the formation of the second emulsion, which ultimatelyforms the MVL particles. In some embodiments, the lysine concentrationin the bupivacaine MVLs produced by the process described herein is atleast about 5%, 10%, 15%, 20%, 25%, or 30% more than the encapsulatedlysine concentration in the Exparel® product manufactured by the currentcommercial process. Since lysine is also a pH modifying agent, the smallchange in lysine concentration also results in the increase of theinternal pH of the bupivacaine MVL particles of about 5.50, as comparedto the internal pH of about 5.34 in a batch of Exparel® productmanufactured by the current commercial process. In some suchembodiments, the increase of the internal pH is characterized by thedecrease in [H⁺] concentration (i.e., pH=− log [H⁺]). In someembodiments, the decrease in [H⁺] concentration in the internal aqueouschambers of the bupivacaine MVLs is at least about 5%, 10%, 15%, 20%,25% or 30%. As reported by Grit M. et al., phospholipids such asphosphatidylcholine have the lowest rate of hydrolysis regardless of thetemperature of their storage at pH 6.5. See J. Pharm. Sci. 1993;82(4):362-366. As such, the closer the internal pH is to 6.5, the lowerthe lipid hydrolysis. In some further embodiments, the dextroseconcentration in the bupivacaine MVLs produced by the process describedherein is at least about 2%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5% or 20%more than the encapsulated dextrose concentration in the Exparel®product manufactured by the current commercial process.

In some embodiments of the composition described herein, the mixing instep (a) is performed using a first mixer at a high shear speed. In someembodiments, the high shear speed is from about 1100 rpm to about 1200rpm, for example, 1100 rpm, 1120 rpm, 1130 rpm, 1140 rpm, 1150 rpm, 1160rpm, 1170 rpm, 1180 rpm, 1190 rpm, or 1200 rpm, or a range defined byany two of the preceding values. In some embodiment, the high shearspeed is about 1150 rpm. In some embodiments, the mixing in step (a) isperformed for about 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes or75 minutes. In some further embodiments, the first mixer used in step(a) of the process is a mixer having a blade diameter of between about 8inch to about 10 inch. In further embodiments, the first mixer used instep (a) of the process is not a static mixer. In further embodiments,the mixing in step (a) is performed at a temperature of about 21° C. toabout 23° C.

In some embodiments of the composition described herein, the mixing instep (b) is performed using a second mixer at a low shear speed. In someembodiments, the low shear speed is from about 450 rpm to about 510 rpm,for example, 450 rpm, 455 rpm, 460 rpm, 465 rpm, 470 rpm, 475 rpm, 480rpm, 485 rpm, 490 rpm, 495 rpm, 500 rpm, 505 rpm, or 510 rpm, or a rangedefined by any of the two preceding values. In some embodiment, the lowshear speed is about 495 rpm. In some embodiments, the mixing in step(b) is performed for about 60 seconds, 61 seconds, 62 seconds, 63seconds, 64 seconds, or 65 seconds. In some further embodiments, thesecond mixer used in step (b) of the process has a blade diameter ofbetween about 10 inch to about 15 inch, for example, 10 inch, 11 inch,12 inch, 13 inch, or 14 inch. In further embodiments, the second mixerused in step (b) of the process is not a static mixer. In furtherembodiments, the mixing in step (b) is performed at a temperature ofabout 21° C. to about 23° C.

In some embodiments of the composition described herein, the compositionof bupivacaine encapsulated multivesicular liposomes may have a finalvolume of about 150 L to about 250 L, or about 200 L to about 250 L,before being filled into individual containers for human administration.In other embodiments, the composition of bupivacaine encapsulated MVLsmay have a volume of 10 mL or 20 mL for a single dose administration. Insome embodiments, the percent packed particle volume (% PPV) of thecomposition of bupivacaine encapsulated MVLs is about 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% or 44%. In some suchembodiments, the concentration of the bupivacaine in the composition isfrom about 12.6 mg/mL to about 17 mg/mL. In one embodiment, theconcentration of the bupivacaine in the composition is about 13.3 mg/mL.In further embodiments, the composition comprises less than 5%, 4%, 3%,2% or 1% unencapsulated bupivacaine, wherein the amount ofunencapsulated bupivacaine is calculated based on the total weight ofthe bupivacaine in the composition. In some embodiments, the d₅₀ of themultivesicular liposomes in the composition is about 24 μm to about 31μm. In one embodiment, the d₅₀ of the multivesicular liposomes in thecomposition is about 27 μm.

Bupivacaine Multivesicular Liposomes

Some aspect of the present disclosure relates to a composition ofbupivacaine encapsulated multivesicular liposomes (MVLs), comprising:bupivacaine residing inside a plurality of internal aqueous chambers ofthe MVLs separated by lipid membranes, wherein the lipid membranescomprise 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid; and an aqueous medium in which the bupivacaineencapsulated MVLs are suspended; wherein erucic acid concentration inthe composition is about 23 μg/mL or less (e.g., about 22.7 μg/mL) afterthe composition is stored at 25° C. for one month. In some embodiments,the composition has an initial pH of about 7.4. In some furtherembodiments, the erucic acid concentration in the composition is about38 μg/mL or less (e.g., about 37.3 μg/mL) after the composition isstored at 25° C. for two months. In some such embodiments, thecomposition has a pH of about 7.1 after the composition is stored at 25°C. for two months. In some further embodiments, the erucic acidconcentration in the composition is about 54 μg/mL or less (e.g., about53.0 μg/mL) after the composition is stored at 25° C. for three months.In some such embodiments, the composition has a pH of about 6.9 afterthe composition is stored at 25° C. for three months. In some furtherembodiments, the erucic acid concentration in the composition is about100 μg/mL or less (e.g., about 98.7 μg/mL) after the composition isstored at 25° C. for six months. In some further embodiments, thecomposition has a pH of about 6.5 after the composition is stored at 25°C. for six months. In some further embodiments, the lipid membranesfurther comprise cholesterol and tricaprylin.

In some embodiments, the erucic acid concentration in the composition ofbupivacaine MVLs is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14% or 15% less than the erucic acid concentrationin the Exparel® product currently on the market, under the sameincubation conditions. In some such embodiments, the incubationcondition is at 25° for 1 month, 2 months, 3 months, or 6 months.

Some additional aspect of the present disclosure relates to acomposition of bupivacaine encapsulated multivesicular liposomes (MVLs),comprising: bupivacaine residing inside a plurality of internal aqueouschambers of the MVLs separated by lipid membranes, wherein the lipidmembranes comprise 1,2-dierucoylphosphatidylcholine (DEPC),1,2-dipalmitoyl-sn-glycero-3 phospho-rac-(1-glycerol) (DPPG), and atleast one neutral lipid; and an aqueous medium in which the bupivacaineencapsulated MVLs are suspended; wherein the internal pH of thebupivacaine encapsulated MVLs is about 5.50. In some embodiments of thecomposition described herein, the internal lysine concentration of thebupivacaine encapsulated MVLs composition is about 0.030 mg/mL to about0.032 mg/mL. In some further embodiments, the internal lysineconcentration of the bupivacaine encapsulated MVLs composition is about0.031 mg/mL. In some embodiments of the composition described herein,the internal dextrose concentration of the bupivacaine encapsulated MVLscomposition is about 1.25 mg/mL to about 1.32 mg/mL. In some furtherembodiments, the internal dextrose concentration of the bupivacaineencapsulated MVLs composition is about 1.29 mg/mL. In some furtherembodiments, the lipid membranes further comprise cholesterol andtricaprylin. In some further embodiments, the internal lysine ordextrose concentration are measured when the bupivacaine MVLs are in anaqueous suspension having % PPV from about 36% to about 38% (e.g., about36.5%).

In some embodiments of the composition described herein, the internallysine concentration in the bupivacaine MVLs is at least about 5%, 10%,15%, 20%, 25%, or 30% more than the encapsulated lysine concentration inthe Exparel® product currently on the market. In some such embodiments,the small change in lysine concentration also results in the increase ofthe internal pH of the bupivacaine MVL particles of about 5.50, ascompared to the internal pH of about 5.34 in the Exparel® productcurrently on the market. In some such embodiments, the increase of theinternal pH is characterized by the decrease in [H⁺] concentration(i.e., pH=− log [H⁺]). In some embodiments, the decrease in [H⁺]concentration in the internal aqueous chambers of the bupivacaine MVLsis at least about 5%, 10%, 15%, 20%, 25% or 30%. In some furtherembodiments, the internal dextrose concentration in the bupivacaine MVLsis at least about 2%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5% or 20% more thanthe encapsulated dextrose concentration in the Exparel® currently on themarket.

In some further embodiments, the composition of bupivacaine encapsulatedMVLs may have a volume of 10 mL or 20 mL for a single doseadministration. In some embodiments, the percent packed particle volume(% PPV) of the composition of bupivacaine encapsulated MVLs is about32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% or 44%. Insome such embodiments, the concentration of the bupivacaine in thecomposition is from about 12.6 mg/mL to about 17 mg/mL. In oneembodiment, the concentration of the bupivacaine in the composition isabout 13.3 mg/mL. In further embodiments, the composition comprises lessthan 5%, 4%, 3%, 2% or 1% unencapsulated bupivacaine, wherein the amountof unencapsulated bupivacaine is calculate based on the total weight ofthe bupivacaine in the composition. In some embodiments, the d₅₀ of themultivesicular liposomes in the composition is about 24 μm to about 31μm. In one embodiment, the d₅₀ of the multivesicular liposomes in thecomposition is about 27 μm.

Methods of Administration

Some embodiments of the present application are related to methods fortreating, ameliorating pain comprising administering a pharmaceuticalcomposition comprising bupivacaine MVLs, as described herein, to asubject in need thereof. In some further embodiments, the pain is postsurgical pain.

In some embodiments of the methods described herein, the administrationis parenteral. In some further embodiments, the parenteraladministration may be selected from the group consisting of subcutaneousinjection, tissue injection, intramuscular injection, intraarticular,spinal injection, intraocular injection, epidural injection, intrathecalinjection, intraotic injection, perineural injection, and combinationsthereof. In particular embodiments, the parenteral administration issubcutaneous injection or tissue injection. In some further embodiments,the instant pharmaceutical compositions can be administered by bolusinjection, e.g., subcutaneous bolus injection, intramuscular bolusinjection, intradermal bolus injection and the like. In one embodiment,the administration is via local infiltration to a surgical site toprovide local analgesia. In another embodiment, the administration isvia interscalene brachial plexus nerve block or femoral nerve block toprovide regional analgesia.

Administration of the instant bupivacaine MVL composition may beaccomplished using standard methods and devices, e.g., pens, injectorsystems, needle and syringe, a subcutaneous injection port deliverysystem, catheters, and the like. The administration of the bupivacaineMVLs composition may be used in conjunction with Pacira's handheldcryoanalgesia device.

Pharmaceutical Compositions

In some embodiments, the composition comprising bupivacaine MVLs is apharmaceutical formulation includes a pharmaceutically acceptablecarrier. Effective injectable bupivacaine MVLs compositions is in aliquid suspension form. Such injectable suspension compositions requirea liquid suspending medium, with or without adjuvants, as a vehicle. Thesuspending medium can be, for example, aqueous solutions of sodiumchloride (i.e., saline solution), dextrose, sucrose,polyvinylpyrrolidone, polyethylene glycol, a pH modifying agentdescribed herein, or combinations of the above. In some embodiments, thesuspending medium of bupivacaine MVLs is a saline solution, optionallycontain a tonicity agent such as dextrose and/or a pH modifying agentsuch as lysine.

Suitable physiologically acceptable storage solution components are usedto keep the compound suspended in suspension compositions. The storagesolution components can be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and the alginates.Many surfactants are also useful as suspending agents. The suspendingmedium could also contain lecithin, alkylphenol polyethylene oxideadducts, naphthalenesulfonates, alkylbenzenesulfonates, or thepolyoxyethylene sorbitan esters. In some embodiments, the bupivacaineMVL composition is free or substantially free of any additive ofpreservatives.

In any embodiments of the composition of bupivacaine encapsulated MVLsdescribed herein, the composition may be a pharmaceutical compositionsuitable for human administration. In further embodiments, thecomposition may be an aqueous suspension of bupivacaine encapsulated MVLparticles.

EXAMPLES

The following examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present application.

Example 1: Lipid Hydrolysis Analysis Based on Erucic Acid Assay

In this example, the lipid stability of three batches (Batch No. 1, 2and 3 in Tables 1A and 1B) of bupivacaine MVLs aqueous suspensionprepared by the new process described herein and were compared to tenreference samples of bupivacaine MVLs aqueous suspension prepared by thecurrent commercial process. DEPC hydrolysis byproduct erucic acid wasused as the marker to measure the stability of the lipid membranes ofthe MVL particles. All the samples were incubated at 25° C. for 1 month,2 months, 3 months and 6 months. The pH of the supernatant of eachsample (i.e., the external pH of the bupivacaine MVL composition) wasalso tested at each time point and summarized in Table 1B. Erucic acidwas detected using HPLC and the erucic acid concentration in the samplewas calculated based on the HPLC peak area and the standard curve.

TABLE 1A Erucic acid concentration in the bupivacaine MVLs as afunctional of time Erucic acid concentration (μg/mL) 1 month 2 months 3months 6 months Batch 1 22 36 54 99 2 23 38 51 99 3 23 38 54 98 Average22.7 37.3 53.0 98.7 % RSD 2.5 3.1 3.3 0.6 Reference samples Average n/a38.7 55.4 113.1 % RSD n/a 24.3 15.0 4.2

TABLE 1B External pH of bupivacaine MVLs compositions as a functional oftime External pH 0 month 1 months 2 months 3 months 6 months Batch 1 7.47.2 7.1 6.9 6.5 2 7.4 7.1 7.1 6.9 6.5 3 7.3 7.1 7.1 6.9 6.5 Average 7.47.1 7.1 6.9 6.5 % RSD 0.8 0.8 0.0 0.0 0.0 Ref. Samples Average 7.1 7.16.9 6.8 6.5 % RSD 1.4 1.6 1.5 0.8 0.6

FIG. 3A is a line chart showing supernatant pH as a function ofincubation time of the bupivacaine-MVL compositions prepared by the newprocess described herein as compared to those prepared by the existingcommercial process. It is known that during incubation at 25° C., theExparel® product pH normally decreases slightly. During the six monthsperiod, the pH of bupivacaine MVL compositions prepared by the presentprocess described herein decreased 37% faster than those prepared by theexisting process, but still within the required 5.8-7.4 pH range.

FIG. 3B is a line chart showing erucic acid concentration as a functionof incubation time at 25° C. of the bupivacaine-MVL compositionsprepared by the new process described herein as compared to thoseprepared by the existing commercial process. It was observed that therate of lipid hydrolysis was 18% lower in the bupivacaine-MVLcompositions prepared by the new process.

FIG. 3C is a line chart showing erucic acid concentration as a functionof supernatant pH at 25° C. Typically, decreases in pH can bothcatalyze, and be a consequence of lipid hydrolysis. Therefore, a morerapid pH decline would normally be associated with a more rapid increasein erucic acid concentration. However, the slope for rate of change oferucic acid concentration as a function of (decreasing) pH was actuallyflatter for the bupivacaine-MVL compositions prepared by the new processas compared to those prepared by the existing commercial process. Theimproved lipid stability (as indicated by the erucic acid concentration)observed in the bupivacaine MVLs prepared by the presently describedprocess was surprisingly unexpected.

Example 2: Measurement of Lysine and Dextrose Concentrations inBupivacaine MVLs

In this experiment, the lysine and dextrose concentration were measuredin three batches (Batch No. 1, 2 and 3 in Tables 2A and 2B) ofbupivacaine MVLs aqueous suspension prepared by the new processdescribed herein and compared to several reference samples ofbupivacaine MVLs aqueous suspension prepared by the current commercialprocess.

TABLE 2A Lysine and dextrose concentrations in bupivacaine MVLcompositions Total Suspension MVL particles Dextrose Lysine DextroseLysine Batch (mg/mL) (mg/mL) (mg/mL) (mg/mL) 1 2.19 0.12 1.32 0.031 22.17 0.12 1.30 0.030 3 2.15 0.12 1.25 0.032 Average 2.17 0.12 1.29 0.031Avg. Ref. Samples 1.86 0.11 1.14 0.024 Avg./Avg. Ref. 1.17 1.08 1.131.29 Samples

TABLE 2B External and internal pH in bupivacaine MVL compositions AvgTime 0 Batch Internal pH Internal pH Sup pH 1 5.49 5.50 7.4 2 5.51 35.50 Ref. samples 5.38 7.1

It was observed that the total suspensions and bupivacaine MVL particlesprepared by the present process contained approximately 17% and 13% moredextrose, respectively, than those samples prepared by the existingcommercial process. In addition, the lysine concentration was 8% and 29%more in those samples prepared by the present process. In addition, theinternal and external pH of the bupivacaine MVL compositions were alsomeasured. The higher internal pH of the bupivacaine MVL particlesprepared by the present process may be attributable to the higher lysineconcentration inside the MVL particles. As discussed above, the slightincrease in MVL internal pH may also contribute to the stability of thelipid membranes.

What is claimed is:
 1. Batches comprising compositions of bupivacaineencapsulated multivesicular liposomes (MVLs) prepared by a process, theprocess of preparing a batch comprising: (a) mixing a first aqueoussolution comprising phosphoric acid with a volatile water-immisciblesolvent solution to form a first water-in-oil emulsion, wherein thevolatile water-immiscible solvent solution comprises bupivacaine, 1,2-dierucoylphosphatidylcholine (DEPC), 1, 2-dipalmitoyl- sn-glycero-3phospho-rac-(1-glycerol) (DPPG), and at least one neutral lipid; (b)mixing the first water-in-oil emulsion with a second aqueous solution toform a second water-in-oil-in-water emulsion, wherein the second aqueoussolution comprises lysine; (c) removing the volatile water-immisciblesolvent from the second water-in-oil-in-water emulsion to form a firstaqueous suspension of bupivacaine encapsulated MVLs having a firstvolume; (d) reducing the first volume of the first aqueous suspension ofbupivacaine encapsulated MVLs by microfiltration to provide a secondaqueous suspension of bupivacaine encapsulated MVLs having a secondvolume; (e) exchanging the aqueous supernatant of the second aqueoussuspension with a saline solution by diafiltration to provide a thirdaqueous suspension of bupivacaine encapsulated MVLs having a thirdvolume; and (f) further reducing the third volume of the third aqueoussuspension by microfiltration to provide a final aqueous suspension ofbupivacaine encapsulated MVLs having a bupivacaine concentration fromabout 11.3 mg/mL to about 17.0 mg/mL; wherein each batch has a volume ofabout 100 L to about 250 L; and wherein an average erucic acidconcentration of at least three batches prepared by the process is about105 μg/mL or less when measured after the compositions are stored at 25°C. for six months.
 2. The batches of claim 1, wherein the average erucicacid concentration of the at least three batches is about 99 μg/mL orless when measured after the compositions are stored at 25° C. for sixmonths.
 3. The batches of claim 1, wherein the average erucic acidconcentration of the at least three batches is greater than about 53μg/mL, when measured after the compositions are stored at 25° C. for sixmonths.
 4. The batches of claim 2, wherein the average erucic acidconcentration of the at least three batches is greater than about 53μg/mL, when measured after the compositions are stored at 25° C. for sixmonths.
 5. The batches of claim 1, wherein the compositions of thebatches have an initial pH of about 7.0 to about 7.4.
 6. The batches ofclaim 1, wherein the compositions of the batches have a pH of about 6.5when measured after the compositions are stored at 25° C. for sixmonths.
 7. The batches of claim 1, wherein the at least one neutrallipid in the volatile water-immiscible solvent solution comprisestricaprylin.
 8. The batches of claim 1, wherein the volatilewater-immiscible solvent solution further comprises cholesterol.
 9. Thebatches of claim 1, wherein the bupivacaine concentration in thecompositions of the batches is about 13.3 mg/mL.
 10. The batches ofclaim 1, wherein the compositions of the batches comprise less than 5%by weight unencapsulated bupivacaine.
 11. The batches of claim 1,wherein the d₅₀ of the MVLs in the compositions of the batches is about24 μm to about 31 μm.
 12. The batches of claim 1, wherein the percentpacked particle volume (% PPV) of the bupivacaine encapsulated MVLs inthe compositions of the batches is about 35% to about 40%.
 13. Thebatches of claim 1, wherein the DEPC and DPPG in the compositions of thebatches are in a mass ratio of about 7:1 to about 10:1.
 14. The batchesof claim 1, wherein the bupivacaine is in a salt form.
 15. The batchesof claim 14, wherein the bupivacaine is in the form of bupivacainephosphate.
 16. The batches of claim 1, wherein the final aqueoussuspension comprises a saline solution.
 17. The batches of claim 1,wherein the mixing in step (a) is performed using a first mixer at ahigh shear speed from about 1100 rpm to about 1200 rpm.
 18. The batchesof claim 17, wherein the high sheer speed is about 1150 rpm.
 19. Thebatches of claim 17, wherein a mixing time in step (a) is about 65 toabout 75 minutes.
 20. The batches of claim 1, wherein the mixing in step(b) is performed using a second mixer at a low shear speed from about450 rpm to about 510 rpm.
 21. The batches of claim 20, wherein the lowshear speed is about 495 rpm.
 22. The batches of claim 20, wherein amixing time in step (b) is about 60 to about 65 seconds.
 23. A method oftreating or ameliorating pain in a subject in need thereof, comprisingadministering a composition of a batch of claim 1 to the subject. 24.The method of claim 23, wherein the administration is via localinfiltration to a surgical site to provide postsurgical local analgesia.25. The method of claim 23, wherein the administration is viainterscalene brachial plexus nerve block or femoral nerve block toprovide postsurgical regional analgesia.
 26. The method of claim 23,wherein the composition has a volume of 10 mL or 20 mL for a single-doseadministration.
 27. The batches of claim 17, wherein the first mixer hasa blade diameter of about 8 inches to about 10 inches.
 28. The batchesof claim 20, wherein the second mixer has a blade diameter of about 10inches to about 15 inches.
 29. The batches of claim 1, wherein thecompositions of the batches comprise lysine encapsulated in the internalaqueous chambers of the MVLs, and the encapsulated lysine concentrationsin the compositions of the batches are at least about 5% higher than theencapsulated lysine concentration of a bupivacaine encapsulated MVLproduct (Exparel®) manufactured by a 45 L commercial process.
 30. Thebatches of claim 29, wherein the encapsulated lysine concentration inthe compositions of the batches is about 0.03 mg/mL.
 31. The batches ofclaim 30, wherein the compositions of the batches have an initial pH ofabout 7.0 to about 7.4.
 32. The batches of claim 31, the compositions ofthe batches have a pH of about 6.5 when measured after the compositionsare stored at 25° C. for six months.
 33. The batches of claim 30,wherein the bupivacaine concentration in the compositions of the batchesis about 13.3 mg/mL.
 34. The batches of claim 30, wherein thecompositions of the batches comprise less than 5% by weightunencapsulated bupivacaine.
 35. The batches of claim 30, wherein themixing in step (a) is performed using a first mixer at a high shearspeed from about 1100 rpm to about 1200 rpm.
 36. The batches of claim35, wherein a mixing time in step (a) is about 65 to about 75 minutes.37. The bathes of claim 35, wherein the first mixer has a blade diameterof about 8 inches to about 10 inches.
 38. The batches of claim 30,wherein the mixing in step (b) is performed using a second mixer at alow shear speed from about 450 rpm to about 510 rpm.
 39. The batches ofclaim 38, wherein a mixing time in step (b) is about 60 to about 65seconds.
 40. The batches of claim 38, wherein the second mixer has ablade diameter of about 10 inches to about 15 inches.
 41. The batches ofclaim 2, wherein the compositions of the batches comprise lysineencapsulated in the internal aqueous chambers of the MVLs, and theencapsulated lysine concentrations in the compositions of the batchesare at least about 5% higher than the encapsulated lysine concentrationof a bupivacaine encapsulated MVL product (Exparel®) manufactured by a45 L commercial process.
 42. The batches of claim 41, wherein theencapsulated lysine concentration in the compositions of the batches isabout 0.03 mg/mL.
 43. The batches of claim 42, wherein the compositionsof the batches have an initial pH of about 7.0 to about 7.4.
 44. Thebatches of claim 43, the compositions of the batches have a pH of about6.5 when measured after the compositions are stored at 25° C. for sixmonths.
 45. The batches of claim 42, wherein the bupivacaineconcentration in the compositions of the batches is about 13.3 mg/mL.46. The batches of claim 42, wherein the compositions of the batchescomprise less than 5% by weight unencapsulated bupivacaine.
 47. Thebatches of claim 42, wherein the mixing in step (a) is performed using afirst mixer at a high shear speed from about 1100 rpm to about 1200 rpm.48. The batches of claim 47, wherein a mixing time in step (a) is about65 to about 75 minutes.
 49. The bathes of claim 47, wherein the firstmixer has a blade diameter of about 8 inches to about 10 inches.
 50. Thebatches of claim 42, wherein the mixing in step (b) is performed using asecond mixer at a low shear speed from about 450 rpm to about 510 rpm.51. The batches of claim 50, wherein a mixing time in step (b) is about60 to about 65 seconds.
 52. The batches of claim 50, wherein the secondmixer has a blade diameter of about 10 inches to about 15 inches.
 53. Amethod of treating or ameliorating pain in a subject in need thereof,comprising administering a composition of a batch of claim 2 to thesubject.
 54. The method of claim 53, wherein the administration is vialocal infiltration to a surgical site to provide postsurgical localanalgesia.
 55. The method of claim 53, wherein the administration is viainterscalene brachial plexus nerve block or femoral nerve block toprovide postsurgical regional analgesia.
 56. The method of claim 53,wherein the composition has a volume of 10 mL or 20 mL for a single-doseadministration.
 57. A method of treating or ameliorating pain in asubject in need thereof, comprising administering a composition of abatch of claim 30 to the subject.
 58. The method of claim 57, whereinthe administration is via local infiltration to a surgical site toprovide postsurgical local analgesia.
 59. The method of claim 57,wherein the administration is via interscalene brachial plexus nerveblock or femoral nerve block to provide postsurgical regional analgesia.60. The method of claim 57, wherein the composition has a volume of 10mL or 20 mL for a single-dose administration.